Methods and compositions for determining bacterial integrity

ABSTRACT

The disclosure provides methods and compositions for determining bacterial integrity.

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 62/435,244, filed Dec. 16, 2016, which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The disclosure provides methods and compositions for determiningbacterial integrity.

BACKGROUND

The human intestinal microbiome includes a large number ofmicroorganisms. A significant number of these microorganisms areanaerobic bacteria. Compositions that include anaerobic bacteria thatoriginated from the human intestinal microbiome have shown potential inthe treatment of human disease (See e.g., Atarashi et al., Nature 500,232, 2013; Atarashi et al., Cell 163, 1, 2015; Mathewson et al., NatureImmunology 17, 505, 2016). However, anaerobic bacteria are challengingto manufacture because of their sensitivity to oxygen. In addition, anyanaerobic bacteria that are to be used for therapeutic applications needto be evaluated for their integrity and readiness for administration.New and improved compositions and methods for determining if anaerobicbacteria have sufficient integrity to be administered are neededtherefore.

SUMMARY

In one aspect, the disclosure provides compositions and methods fordetermining bacterial integrity.

In some embodiments, the disclosure provides a method for determiningthe integrity of a bacterial composition, the method comprising: growingthe bacterial composition on a selective medium, wherein the bacterialcomposition comprises a bacterial strain, and wherein if the bacterialcomposition grows slower on the selective medium than on a non-selectivemedium, the integrity of the bacterial composition is compromised. Insome embodiments of the methods provided herein, the method furthercomprises growing the bacterial composition on the non-selective medium.In some embodiments, the bacterial composition was lyophilized prior togrowing the bacterial composition. In some embodiments, if the integrityof the bacterial composition is compromised, the bacterial compositionis not used to prepare a live bacterial product.

In some embodiments, the disclosure provides a method for determiningthe integrity of a first bacterial composition, the method comprising:growing the first bacterial composition on a selective medium, whereinthe first bacterial composition comprises a bacterial strain; growing asecond bacterial composition on the selective medium, wherein the secondbacterial composition comprises a bacterial strain, and wherein if thefirst bacterial composition grows slower than the second bacterialcomposition, the integrity of the first bacterial composition iscompromised.

In some embodiments, the disclosure provides a method for determiningthe integrity of a first bacterial composition, the method comprising:growing the first bacterial composition on a selective medium, growingthe first bacterial composition on a non-selective medium, wherein thefirst bacterial composition comprises a bacterial strain; growing asecond bacterial composition on the selective medium, growing the secondbacterial composition on the non-selective medium, wherein the secondbacterial composition comprises a bacterial strain; wherein if thedifference in growth between the selective medium and the non-selectivemedium for the first bacterial composition is greater than thedifference in growth between the selective medium and the non-selectivemedium for the second bacterial composition, and both the firstbacterial composition and the second bacterial composition grow sloweron the selective medium than the non-selective medium, the integrity ofthe first bacterial composition is compromised.

In some embodiments of any of the methods provided herein, the bacterialcomposition was lyophilized prior to growing the bacterial composition.In some embodiments of any of the methods provided herein, the firstbacterial composition was lyophilized prior to growing the firstbacterial composition. In some embodiments of any of the methodsprovided herein, the second bacterial composition was lyophilized priorto growing the second bacterial composition.

In some embodiments of any of the methods provided herein, the selectivemedium is bile acid media.

In some embodiments, if the integrity of the first bacterial compositionis compromised, the first bacterial composition Is not used to prepare alive bacterial product.

In some embodiments of any of the methods provided herein, the firstbacterial composition and the second bacterial composition comprise thesame bacterial strain. In some embodiments of any of the methodsprovided herein, the bacterial strain is an anaerobic bacterial strain.In some embodiments, the bacterial strain belongs to the classClostridia. In some embodiments, the bacterial strain belongs to thefamily Clostridiaceae. In some embodiments, the bacterial strain belongsto the genus Clostridium. In some embodiments, the bacterial strain isselected from the group consisting of Clostridium bolteae, Anaerotruncuscolihominis, Ruminococcus torques, Clostridium symbiosum, Blautiaproducta, Dorea longicatena, Erysipelotrichaceae bacterium, andSubdolinogranulum spp. In some embodiments of any of the methodsprovided herein, the bacterial strain is Clostridium bolteae. In someembodiments of any of the methods provided herein, the bacterial strainis Dorea longicatena.

In some embodiments, the bacterial strain comprises a 16S rDNA sequencehaving at least 97% sequence identity with a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 1-8.

These and other aspects of the invention, as well as various embodimentsthereof, will become more apparent in reference to the drawings anddetailed description of the invention.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. Thefigures are illustrative only and are not required for enablement of thedisclosure. For purposes of clarity, not every component may be labeledin every drawing. In the drawings:

FIGS. 1A and 1B show the results of experiments with Dorea longicatena(Strain 6). In FIG. 1A, the bars from left to right are: TSA blood(Fresh), TSA blood+bile (Fresh), TSA blood (Frozen), TSA blood+bile(Frozen), TSA blood (Lyo), TSA blood+bile (Lyo). In FIG. 1B, the barsfrom left to right are: TSA blood (Fresh), TSA blood+bile (Fresh), TSAblood (Frozen), TSA blood+bile (Frozen), TSA blood (Lyo), TSA blood+bile(Lyo). TSA: Tryptic Soy Agar. Lyo: lyophilized.

FIGS. 2A and 2B show the results of experiments with Clostridium bolteae(Strain 1). In FIG. 2A, the bars from left to right are: PYG (Fresh),TSA blood (Fresh), TSA blood and ox bile (Fresh), PYG (Lyo), TSA blood(Lyo), TSA blood and ox bile (Lyo). In FIG. 2B, the bars from left toright are: TSA blood (Fresh), TSA blood and ox bile (Fresh), TSA blood(Lyo), TSA blood and ox bile (Lyo). PYG: Peptone Yeast Glucose media.TSA: Tryptic Soy Agar. Lyo: lyophilized.

FIGS. 3A and 3B show the results of experiments assessing the viabilityof the indicated bacterial strains. FIG. 3A shows bacterial viabilityprior to lyophilization and storage at −80° C. FIG. 3B shows bacterialviability after lyophilization and storage at −80° C. In FIG. 3A, thebars for each of the strains are left: viability on chocolate agar, andright: viability on chocolate agar+ox bile. In FIG. 3B, the bars foreach of the strains are left: viability on chocolate agar, and right:viability on chocolate agar+ox bile.

FIGS. 4A and 4B show the results of experiments assessing the viabilityof bacterial strains following storage at 25° C. FIG. 4A shows bacterialviability following short term storage at 25° C. FIG. 4B shows bacterialviability after one month of storage at 25° C. In FIG. 4A, the bars foreach of the strains are, from left to right, initial viability (T0Viable Titer cfu/0.1 g); initial viability in medium with ox bile (T0Titer on Ox Bile); viability after 48 hours (48 hr Viable Titer cfu/0.1g); viability after 48 hours with ox bile (48 hr Titer on Ox Bile);viability after one week (1 week Viable Titer cfu/0.1 g); and viabilityafter one week with ox bile (1 week Titer on Ox Bile). In FIG. 4B, thebars for each of the strains are, from left to right, initial viability(T0 Viable Titer cfu/0.1 g); initial viability in medium with ox bile(T0 Titer on Ox Bile); viability after one month (1 mo Viable Titercfu/0.1 g); and viability after one month with ox bile (1 mo Titer on OxBile).

DETAILED DESCRIPTION

The preparation and preservation of bacterial compositions fortherapeutic use, in particular anaerobic bacteria, has been challenging.While bacteria can be frozen down and regrown on plates or in solution,it has been difficult to standardize this process. There is a need topreserve bacteria that can be used for therapeutic purposes.Lyophilization is a recognized process for the preservation of peptidesand proteins. However, lyophilization of bacterial compositions, inparticular anaerobic bacteria, has been challenging, and loss ofintegrity has been found upon the lyophilization of bacterialcompositions.

It is relatively straightforward to distinguish between viable andnon-viable bacteria. Viable bacteria can divide and cultures can begenerated, while non-viable bacteria can no longer be grown up (do notreplicate and cannot be cultured). The challenge lies in distinguishingviable bacteria from bacteria that have lost integrity. Bacteria thathave lost integrity can still be grown up, meaning the bacterial cellsare viable and culturable. However, they will grow more slowly thanhealthy bacteria.

Additionally, bacteria that have lost integrity may no longer besuitable for use in pharmaceutical compositions. For example,pharmaceutical compositions that transit through the digestive tractwhen administered to a subject may be exposed to the acidic pH of thestomach. If the active agent(s) of the pharmaceutical composition areintended to reach the intestine and may become exposed to stomach acid,the agent must survive the conditions in the stomach to reach theappropriate site. In addition, if the active agent in the pharmaceuticalcomposition that is targeted for the intestine is a bacterial strain,that bacterial strain must be able to grow in, or at least be able tosurvive, the environment of the intestine for a sufficient time to beeffective.

The methods described herein allow for the assessment of the integrityof bacterial compositions which may be used to produce pharmaceuticalcompositions, for example as part of a quality control process duringthe manufacturing of pharmaceutical compositions. If the bacterialcompositions are found to lack integrity or have compromised integrity,the bacterial compositions are not used in a pharmaceutical composition.Thus, for instance if pharmaceutical composition comprises one or morebacterial compositions (e.g., if the pharmaceutical composition is acocktail of bacterial strains), each of those bacterial compositions istested for integrity. If the integrity of a particular bacterialcomposition is found to be insufficient, the tested bacterialcomposition will not be included in the pharmaceutical composition. If aparticular bacterial composition is required in a pharmaceuticalcomposition, additional batches of the pharmaceutical composition may begenerated and evaluated for integrity. A batch of the particularbacterial composition that has sufficient integrity can then be used togenerate the pharmaceutical composition.

Furthermore, pharmaceutical compositions must withstand storageconditions prior to administration to subjects. Bacterial compositionlacking integrity or having compromised integrity may not survivestorage conditions and therefore, may be less effective uponadministration.

It was surprisingly found herein that selective media can be used todistinguish healthy bacterial compositions from bacterial compositionsthat have lost integrity. As shown herein, both healthy bacterialcompositions and bacterial compositions that have lost integrity willgrow at a similar rate on non-selective media. Surprisingly, bacterialcompositions that have lost integrity were found to have a much slowergrowth rate on selective media than healthy bacterial compositions.Bacterial compositions as used herein refer to a composition thatincludes bacteria, such as any of the bacterial strains describedherein.

In one aspect, the disclosure provides a method for determining theintegrity of a bacterial composition, wherein the method includes a stepof growing the bacterial composition on a selective medium. If thebacterial composition grows slower on the selective medium than on anon-selective medium, the integrity of the bacterial composition iscompromised. Alternatively, the disclosure provides a method fordetermining the integrity of a bacterial composition, wherein the methodincludes a step of growing the bacterial composition on a selectivemedium. If the bacterial composition grows similarly on the selectivemedium as on a non-selective medium, the integrity of the bacterialcomposition is not compromised. The bacterial growth can be measuredagainst a previously obtained standard growth curve for eithernon-selective or selective media (e.g., a reference growth curve), orthe bacterial composition can be grown on both selective medium andnon-selective medium in the same evaluations.

In one aspect, the disclosure provides a method for determining theintegrity of a first bacterial composition, wherein the method includes:growing the first bacterial composition on a selective medium, growing asecond bacterial composition on the selective medium, wherein if thefirst bacterial composition grows slower than the second bacterialcomposition, the integrity of the first bacterial composition iscompromised. Alternatively, the disclosure provides a method fordetermining the integrity of a first bacterial composition, wherein themethod includes: growing the first bacterial composition on a selectivemedium, growing a second bacterial composition on the selective medium,wherein if the first bacterial composition grows at as similar rate asthe second bacterial composition, the integrity of the first bacterialcomposition is not compromised. In some embodiments, the first andsecond bacterial composition include the same bacterial composition buthave been prepared differently. Thus, for instance, a first bacterialcomposition was prepared fresh, while the second bacterial compositionis a lyophilized bacterial composition. Generally, the first and secondbacterial composition will include the same bacterial strain. However,in some embodiments, related strains (e.g., strains from the samebacterial species) are used in the first and second bacterialcomposition.

In one aspect, the disclosure provides a method for determining theintegrity of a first bacterial composition, wherein the method includesgrowing the first bacterial composition on a selective medium and on anon-selective medium, growing a second bacterial composition on theselective medium and a non-selective medium, wherein if the differencein growth between the selective medium and the non-selective medium forthe first bacterial composition is greater than the difference in growthbetween the selective medium and the non-selective medium for the secondbacterial composition, and both the first bacterial composition and thesecond bacterial composition grow slower on the selective medium thanthe non-selective medium, the integrity of the first bacterialcomposition is compromised. Alternatively, the disclosure provides amethod for determining the integrity of a first bacterial composition,wherein the method includes growing the first bacterial composition on aselective medium and on a non-selective medium, growing a secondbacterial composition on the selective medium and a non-selectivemedium, wherein if the difference in growth between the selective mediumand the non-selective medium for the first bacterial composition issimilar to the difference in growth between the selective medium and thenon-selective medium for the second bacterial composition, the integrityof the first bacterial composition is not compromised.

As will be appreciated by one of ordinary skill in the art, the growthof bacteria in a particular medium (e.g., selective, non-selective) maybe assessed by any method known in the art. For example, in someembodiments, growth of bacteria in a medium may be assessed byquantifying the colony forming units (cfus). In some embodiments, thegrowth of bacteria in a medium may be assessed by measuring the opticaldensity of the bacterial culture (e.g., OD₆₀₀), for example over time(e.g., growth curve) or as a maximal density achieved by the culture.

It should be appreciated that the methods disclosed herein can be usedas part of quality control. As used herein, the term “quality control”refers to a process or step used to assess the quality of a component,such as a bacterial composition, in the process of producing a product.If the quality of the component meets particular criteria, the componentmay be used, for example to produce a product; whereas if the quality ofthe component fails to meet the criteria, the component is not used. Insome embodiments, a bacterial composition may be subjected to qualitycontrol to assess the bacterial integrity according any of the methodsdescribed herein.

In some embodiments, a bacterial composition can undergo one or moresteps in a manufacturing process and/or a step to prepare the bacterialcompositions for use in a therapeutic (pharmaceutical) composition. Suchsteps include lyophilization, formulation, drying, equilibration, etc.After the bacterial composition has undergone one or more of themanufacturing/preparation steps, the bacterial composition may beevaluated for bacterial integrity according to the methods providedherein. In some embodiments, a bacterial composition is lyophilized, andthe lyophilized bacterial composition is evaluated for loss ofintegrity. If the bacterial composition is found to have integrity(i.e., was not found to have a loss of integrity), the bacterialcomposition can be used for further manufacturing and/or preparationsteps, for example to produce a pharmaceutical composition. Bacterialcompositions found to have integrity (i.e., not found to have a loss ofintegrity) may be considered to have passed quality control and may beused, for example, in one or more further manufacturing and/orpreparation steps (e.g., to produce a product such as a pharmaceuticalcomposition). If the lyophilized bacterial composition is found not tohave lost integrity, it is used to produce pharmaceutical compositions.If the bacterial composition is found to have a loss of integrity, thebacterial composition will not be used for further manufacturing and/orpreparation steps. Bacterial compositions found to have a loss ofintegrity may be considered to have failed quality control and are notused, for example, in one or more further manufacturing and/orpreparation steps (e.g., not used to produce a product such as apharmaceutical composition). If the lyophilized bacterial composition isfound to have a loss of integrity, it is not used in pharmaceuticalcompositions.

In some embodiments, bacterial compositions may be subjected to storageconditions for a period of time. In some embodiments, bacterialcompositions that have a loss of integrity or have reduced integrity maynot remain viable following storage for a period of time. In someembodiments, if a bacterial composition is found to have a loss ofintegrity, it is not used to product a product that will be subjected tostorage conditions.

In some embodiments, the disclosure provides methods of quality controlof a pharmaceutical composition comprising multiple bacterial strains.In some embodiments, the methods of quality control comprise growing upeach of the bacterial strains of the pharmaceutical compositionsseparately, followed by lyophilization resulting in a bacterialcomposition of each of the bacterial strains of the pharmaceuticalcompositions. Optionally, each of the bacterial compositions may bestored for a prolonged period of time. Each of the bacterialcompositions is subsequently tested in one or more of the methods fordetermining bacterial integrity described herein, and will only beincluded in the pharmaceutical composition if it has sufficientintegrity.

Provided herein are compositions and methods for determining bacterialintegrity. Bacterial integrity as used herein generally refers to thehealth and viability of bacteria and the ability to use the bacteria inmanufacturing methods and/or therapeutic compositions (pharmaceuticalcompositions). In some embodiments, bacteria that do not have sufficientintegrity are bacteria that grow slower than healthy bacteria. Thesebacteria with insufficient integrity, for instance, have a longer lagphase than healthy bacteria. Loss of integrity may be correlated withinjury to the bacteria, for instance, the bacteria may have a loss ofmembrane integrity, loss of cell wall integrity, protein denaturation,cross-linking, chemical modifications, and/or DNA damage. In someembodiments, a bacterial composition is considered to have a loss ofintegrity or have compromised integrity if the bacterial composition hasa slower growth rate as compared to another bacterial composition. Insome embodiments, the bacterial composition having a loss of integrityor compromised integrity has a slower growth rate than the samebacterial composition (e.g., same bacterial strain(s)) grown underdifferent conditions (e.g., with non-selective medium). In someembodiments, the bacterial composition having a loss of integrity orcompromised integrity has a slower growth rate than another bacterialcomposition (e.g., containing one or more different bacterial strain(s))grown under the same conditions (e.g., with selective medium).

In some embodiments, a bacterial composition is considered to have aloss of integrity or have compromised integrity if the bacterialcomposition has a growth rate (e.g., doubling time) that is longer thanthe growth rate (e.g., doubling time) of another bacterial compositionby at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75% or more. In some embodiments, growth rate of the bacterialcomposition having a loss of integrity or compromised integrity isslower than the growth rate of the same bacterial composition (e.g.,same bacterial strain(s)) grown under different conditions (e.g., withnon-selective medium). In some embodiments, the growth rate of thebacterial composition having a loss of integrity or compromisedintegrity is slower than the growth rate of another bacterialcomposition (e.g., containing one or more different bacterial strain(s))grown under the same conditions (e.g., with selective medium).

In some embodiments, a bacterial composition is considered to have aloss of integrity or to have compromised integrity if the bacterialcomposition has fewer viable bacteria when grown in selective medium, ascompared to the bacterial composition grown in non-selective medium. Insome embodiments, a bacterial composition is considered to have a lossof integrity or to have compromised integrity if the bacterialcomposition has fewer viable bacteria when grown in selective medium, ascompared to another bacterial composition grown in selective medium. Insome embodiments, a bacterial composition is considered to have a lossof integrity or to have compromised integrity if the bacterialcomposition has a reduction in the viable bacteria by at least 5-fold,10-fold, 100-fold, 1000-fold, 10⁴-fold, 10⁵-fold or more when grown inselective medium, as compared to the bacterial composition grown innon-selective medium or compared to another bacterial composition grownin selective medium.

In some embodiments of the methods provided herein, the bacterialcompositions are grown on selective media. As used herein, the term“selective media” and “selective medium” may be used interchangeably andrefer to any growth medium that allows for differentiation betweenbacterial integrity. In some embodiment, the selective medium contains aselective component, such as a stress-inducing agent. In someembodiments, the stress-inducing agent is bile acid. In some embodimentsof the methods provided herein, the selective medium is bile acid media.The methods disclosed herein, include the element of the use ofselective media to distinguish between bacterial compositions that haveintegrity and bacterial compositions that do not have integrity.Selective media are known in the art and allow for the growth ofselective microorganisms. As disclosed herein, it was unexpected thatselective media can be used to distinguish between bacteria that do ordo not have integrity, rather than distinguishing between differentbacterial strains, which is the traditional use of selective media.

In general, growth on a bile acid, such as ox bile, measures theviability of the bacterial composition under the stress condition ofexposure to bile salts. Bile has antimicrobial properties and can affectthe cell membrane, macromolecule stability, and can cause DNA damage inbacterial cells. Assessing bacterial growth in the presence of bile acidrelative to the bacterial growth in the absence of bile acid allows fora comparison of bacterial strains under a stress condition thatsimulates conditions of the human gastrointestinal tract (see, e.g.,Begley et al. FEMS Microbiol. Rev (2005) 29:625-651). In someembodiments of the methods provided herein, the selective medium is bileacid media. Bile acids include both primary and secondary bile acids andmay be obtained from any source known in the art. Examples of bile acidsare taurocholic acid (a derivative of cholic acid), glycocholic acid (aderivative of cholic acid), taurochenodeoxycholic acid (a derivative ofchenodeoxycholic acid), and glycochenodeoxycholic acid (a derivative ofchenodeoxycholic acid). In some embodiments of the methods providedherein, bile acid media is Ox Bile.

In some embodiments of the methods provided herein, the bacterialcompositions are grown on non-selective media. As used herein, the terms“non-selective media” and “non-selective medium” may be usedinterchangeably. Examples of non-selective medium are known in the art.In some embodiments, the non-selective medium may be the same medium asthe selective medium without a selective agent. For example, in someembodiments, the selective medium is chocolate medium with ox bile andthe non-selective medium is chocolate medium without ox bile.

As will be appreciated by one of skill in the art, a selective mediumand/or a non-selective medium may be in liquid (e.g., broth) or in solidform (e.g., agar).

The methods used herein can be assessed to determine the bacterialintegrity of any bacterial composition. In some embodiments, thebacterial composition has undergone one or more manufacturing steps toprepare it for use in a therapeutic composition. In some embodiments,one or more of the bacterial compositions disclosed herein (e.g., thefirst bacterial composition or the second bacterial composition) waslyophilized prior to being used in the methods provided herein (e.g.,prior to being grown on selective or non-selective media). Methods oflyophilizing compositions, including compositions comprising bacteria,are known in the art. See, e.g., U.S. Pat. Nos. 3,261,761; 4,205,132;PCT Publications WO 2014/029578, WO 2012/098358, WO2012/076665 andWO2012/088261, herein incorporated by reference in their entirety.

The methods provided herein can be used to evaluate the bacterialintegrity of any bacterial strain. Bacterial strains that can be used inthe methods provided herein include aerobic bacteria, anaerobic bacteriaincluding both facultative anaerobes and obligate or strict anaerobes.

In some embodiments of any of the methods provided herein, the firstbacterial composition and the second bacterial composition comprise thesame bacterial strain.

In some embodiments of any of the methods provided herein, the bacterialstrain is an anaerobic bacterial strain. In some embodiments of any ofthe methods provided herein, the bacterial strain is Clostridiumbolteae. In some embodiments of any of the methods provided herein, thebacterial strain is Dorea longicatena.

In some embodiments, the composition includes one or more bacterialstrains. In some embodiments of the compositions provided herein, thebacteria are anaerobic bacteria. In some embodiments of the compositionsprovided herein, the anaerobic bacteria are strict anaerobic bacteria.In some embodiments of the compositions provided herein, the bacteriaare from the class Clostridia. In some embodiments of the compositionsprovided herein, the bacteria are from the family Clostridiaceae. Insome embodiments of the compositions provided herein, the bacteria arefrom the genus Clostridium. In some embodiments of the compositionsprovided herein, the bacteria belong to Clostridium cluster IV, XIVa,XVI, XVII, or XVIII. In some embodiments of the compositions providedherein, the bacteria belong to Clostridium cluster IV, XIVa, or XVII. Insome embodiments of the compositions provided herein, the bacteriabelong to Clostridium cluster IV or XIVa.

In some embodiments of the compositions provided herein, the compositionincludes one or more of the following bacterial strains: Clostridiumbolteae, Anaerotruncus colihominis, Eubacterium fissicatena, Clostridiumsymbiosum, Blautia producta, Dorea longicatena, Erysipelotrichaceaebacterium and Subdolinogranulum spp. In some embodiments of thecompositions provided herein, the composition includes one or more ofthe following bacterial strains: Clostridium bolteae 90A9, Anaerotruncuscolihominis DSM17241, Sellimonas intestinalis, Clostridium bacteriumUC5.1-1D4, Dorea longicatena CAG:42, Erysipelotrichaceae bacterium 21-3,and Clostridium orbiscindens 1_3_50AFAA. In some embodiments of thecompositions provided herein, the composition includes two or more(e.g., 2, 3, 4, 5, 6, 7, or 8) of the following bacterial strains:Clostridium bolteae, Anaerotruncus colihominis, Eubacterium fissicatena,Clostridium symbiosum, Blautia producta, Dorea longicatena,Erysipelotrichaceae bacterium, and Subdolinogranulum spp. In someembodiments, the composition includes Clostridium bolteae. In someembodiments, the composition includes Anaerotruncus colihominis. In someembodiments, the composition includes Eubacterium fissicatena. In someembodiments, the composition includes Clostridium symbiosum. In someembodiments, the composition includes Blautia producta. In someembodiments, the composition includes Dorea longicatena. In someembodiments, the composition includes Erysipelotrichaceae bacterium. Insome embodiments, the composition includes Subdolinogranulum spp.

In one aspect, as shown herein (e.g., in the Examples) the methodsdescribed herein allow for the determination of bacterial integrity. Inone aspect, as shown herein (e.g., in the Examples) the methods providedherein allow for the determination of bacterial integrity of bacterialstrains belonging to Clostridium cluster IV, XIVa, or XVII. In oneaspect, as shown herein (e.g., in the Examples) the methods providedherein allow for the determination of bacterial integrity of any of theanaerobic bacterial strains Clostridium bolteae, Anaerotruncuscolihominis, Eubacterium fissicatena, Clostridium symbiosum, Blautiaproducta, Dorea longicatena, Erysipelotrichaceae bacterium and/orSubdolinogranulum spp. The exemplary bacterial strains of compositionsdisclosed herein can also be identified by their 16S rRNA sequences (SEQID NOs: 1-8). Identifying bacteria by their sequences furthermore allowsfor the identification of additional bacterial strains that areidentical or highly similar to the exemplified bacteria. For instance,the 16S rRNA sequences of bacterial strains were used to identify theclosest relative (based on percent identity) through whole genomesequencing and by comparing these sequences with 16S databases (Table1). In addition, based on whole genome sequencing and comparing of thewhole genome to whole genome databases, the bacterial strains having 16SrRNA sequences provided by SEQ ID NOs: 1-8 are most closely related tothe following bacterial species: Clostridium bolteae 90A9, Anaerotruncuscolihominis DSM 17241, Dracourtella massiliensis GD1, Clostridiumsymbiosum WAL-14163, Clostridium bacterium UC5.1-1D4, Dorea longicatenaCAG:42, Erysipelotrichaceae bacterium 21_3, and Clostridium orbiscindens1_3_50AFAA (see, e.g., Table 1). Thus, in one aspect it should beappreciated that each row of Table 1, the bacterial strains are highlysimilar and/or are identical. In some embodiments, in context of theinstant disclosure the names of bacterial strains within a row of Table1 can be used interchangeably.

TABLE 1 Examples of bacterial species for use in the methods andcompositions disclosed herein Closest species based on SEQ Closestspecies based on Consensus SEQ ID # of 16S Closest species based onStrain ID Sanger sequencing of region as compared with 16S WGS comparedversus Additional closely Clostridium number NO: 16S region database WGdatabases related sequences cluster 1 1 Clostridium bolteae Clostridiumbolteae Clostridium bolteae 90A9 XIVa 2 6 Anaerotruncus Anaerotruncuscolihominis Anaerotruncus IV colihominis colihominis DSM 17241 3 3Eubacterium fissicatena Dracourtella massiliensis Dracourtellamassiliensis Ruminococcus XIVa GD1 torques; Sellimonas intestinalis 4 4Clostridium symbiosum Clostridium symbiosum Clostridium symbiosum XIVaWAL-14163 5 5 Blautia producta Blautia producta Clostridium bacteriumBlautia producta XIVa UC5.1-1D4 ATCC 27340 6 2 Dorea longicatena Dorealongicatena Dorea longicatena CAG:42 XIVa 7 7 Clostridium innocuumClostridium innocuum Erysipelotrichaceae XVIII bacterium 21_3 8 8Flavinofractor plautii Flavinofractor plautii Clostridium orbiscindensSubdolinogranulum IV 1_3_50AFAA

Aspects of the disclosure relate to bacterial strains with 16S rDNAsequences that have a level of sequence identity to a nucleic acidsequence of any one of the sequences of the bacterial strains or speciesdescribed herein. Two or more sequences may be assessed for the identitybetween the sequences. The terms “identical,” percent “identity” in thecontext of two or more nucleic acids or amino acid sequences, refer totwo or more sequences or subsequences that are the same. Two sequencesare “substantially identical” if two sequences have a specifiedpercentage of amino acid residues or nucleotides that are the same(e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%,99.7%, 99.8% or 99.9% sequence identity) over a specified region of anucleic acid or amino acid sequence or over an entire sequence, whencompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Optionally, the identity exists over a region that is atleast about 50 nucleotides in length, or more preferably over a regionthat is 100 to 500 or 1000 or more nucleotides in length. In someembodiments, the identity exists over the length the 16S rRNA or 16SrDNA sequence.

In some embodiments, the bacterial composition includes one or morebacterial strains, wherein the one or more bacterial strains include oneor more 16S rDNA sequences having at least 97% sequence identity withnucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Insome embodiments, the bacterial composition includes one bacterialstrain, wherein the bacterial strain includes one or more 16S rDNAsequences having at least 97% sequence identity with nucleic acidsequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments,the bacterial composition includes one bacterial strain, wherein thebacterial strain includes one or more 16S rDNA sequences having at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, at least 99.9%, or up to 100% sequence identity with nucleic acidsequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

In some embodiments, the bacterial composition includes one bacterialstrain, wherein the bacterial strain includes one or more 16S rDNAsequences having at least 97% sequence identity with the nucleic acidsequence of SEQ ID NO: 1. In some embodiments, the bacterial compositionincludes one bacterial strain, wherein the bacterial strain includes oneor more 16S rDNA sequences having at least 97% sequence identity withthe nucleic acid sequence of SEQ ID NO: 2. In some embodiments, thebacterial composition includes one bacterial strain, wherein thebacterial strain includes one or more 16S rDNA sequences having at least97% sequence identity with the nucleic acid sequence of SEQ ID NO: 3. Insome embodiments, the bacterial composition includes one bacterialstrain, wherein the bacterial strain includes one or more 16S rDNAsequences having at least 97% sequence identity with the nucleic acidsequence of SEQ ID NO:4. In some embodiments, the bacterial compositionincludes one bacterial strain, wherein the bacterial strain includes oneor more 16S rDNA sequences having at least 97% sequence identity withthe nucleic acid sequence of SEQ ID NO:5. In some embodiments, thebacterial composition includes one bacterial strain, wherein thebacterial strain includes one or more 16S rDNA sequences having at least97% sequence identity with the nucleic acid sequence of SEQ ID NO:6. Insome embodiments, the bacterial composition includes one bacterialstrain, wherein the bacterial strain includes one or more 16S rDNAsequences having at least 97% sequence identity with the nucleic acidsequence of SEQ ID NO:7. In some embodiments, the bacterial compositionincludes one bacterial strain, wherein the bacterial strain includes oneor more 16S rDNA sequences having at least 97% sequence identity withthe nucleic acid sequence of SEQ ID NO:8.

Aspects of the disclosure relate to bacterial strains with 16S rDNAsequences that have homology to a nucleic acid sequence of any one ofthe sequences of the bacterial strains or species described herein. Insome embodiments, the bacterial strain has at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% homology relative to anyof the strains or bacterial species described herein over a specifiedregion of nucleic acid or amino acid sequence or over the entiresequence. It would be appreciated by one of skill in the art that theterm “homology” or “percent homology,” in the context of two or morenucleic acid sequences or amino acid sequences, refers to a measure ofsimilarity between two or more sequences or portion(s) thereof. Thehomology may exist over a region of a sequence that is at least about 50nucleotides in length, or more preferably over a region that is 100 to500 or 1000 or more nucleotides in length. In some embodiments, thehomology exists over the length the 16S rRNA or 16S rDNA sequence, or aportion thereof.

In some embodiments, the bacterial composition includes one or morebacterial strains, wherein the one or more bacterial strains include oneor more 16S rDNA sequences having at least 97% homology with nucleicacid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In someembodiments, the bacterial composition includes one bacterial strain,wherein the bacterial strain includes one or more 16S rDNA sequenceshaving at least 97% homology with nucleic acid sequences SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, or SEQ ID NO:8. In some embodiments, the bacterial compositionincludes one bacterial strain, wherein the bacterial strain includes oneor more 16S rDNA sequences having at least 80%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or upto 100% homology with nucleic acid sequences SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQID NO:8.

In some embodiments, the bacterial composition includes one bacterialstrain, wherein the bacterial strain includes one or more 16S rDNAsequences having at least 97% homology with the nucleic acid sequence ofSEQ ID NO:1. In some embodiments, the bacterial composition includes onebacterial strain, wherein the bacterial strain includes one or more 16SrDNA sequences having at least 97% homology with the nucleic acidsequence of SEQ ID NO:2. In some embodiments, the bacterial compositionincludes one bacterial strain, wherein the bacterial strain includes oneor more 16S rDNA sequences having at least 97% homology with the nucleicacid sequence of SEQ ID NO:3. In some embodiments, the bacterialcomposition includes one bacterial strain, wherein the bacterial strainincludes one or more 16S rDNA sequences having at least 97% homologywith the nucleic acid sequence of SEQ ID NO:4. In some embodiments, thebacterial composition includes one bacterial strain, wherein thebacterial strain includes one or more 16S rDNA sequences having at least97% homology with the nucleic acid sequence of SEQ ID NO:5. In someembodiments, the bacterial composition includes one bacterial strain,wherein the bacterial strain includes one or more 16S rDNA sequenceshaving at least 97% homology with the nucleic acid sequence of SEQ IDNO:6. In some embodiments, the bacterial composition includes onebacterial strain, wherein the bacterial strain includes one or more 16SrDNA sequences having at least 97% homology with the nucleic acidsequence of SEQ ID NO:7. In some embodiments, the bacterial compositionincludes one bacterial strain, wherein the bacterial strain includes oneor more 16S rDNA sequences having at least 97% homology with the nucleicacid sequence of SEQ ID NO:8.

In some embodiments, the composition includes a bacterial strain thatincludes a 16S RNA nucleic acid sequence with at least 97% sequenceidentity with the 16S RNA sequence of SEQ ID NO:1. In some embodiments,the composition includes a bacterial strain that includes a 16S RNAnucleic acid sequence with at least 97% sequence identity with the 16SRNA sequence of SEQ ID NO:2.

Additionally, or alternatively, two or more sequences may be assessedfor the alignment between the sequences. The terms “alignment” orpercent “alignment” in the context of two or more nucleic acids or aminoacid sequences, refer to two or more sequences or subsequences that arethe same. Two sequences are “substantially aligned” if two sequenceshave a specified percentage of amino acid residues or nucleotides thatare the same (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical) over a specified regionof the nucleic acid or amino acid sequence or over the entire sequence,when compared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Optionally, the alignment exists over a region that is atleast about 50 nucleotides in length, or more preferably over a regionthat is 100 to 500 or 1000 or more nucleotides in length. In someembodiments, the identity exists over the length the 16S rRNA or 16SrDNA sequence.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. Methods of alignment ofsequences for comparison are well known in the art. See, e.g., by thelocal homology algorithm of Smith and Waterman (1970) Adv. Appl. Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch, J.Mol. Biol. (1970) 48:443, by the search for similarity method of Pearsonand Lipman. Proc. Natl. Acad. Sci. USA (1998) 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group.Madison. Wis.), or by manual alignment and visual inspection (see. e.g.,Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons,Inc. (Ringbou ed., 2003)). Two examples of algorithms that are suitablefor determining percent sequence identity and sequence similarity arethe BLAST and BLAST 2.0 algorithms, which are described in Altschul etal., Nuc. Acids Res. (1977) 25:3389-3402, and Altschul et al., J. Mol.Biol. (1990) 215:403-410, respectively.

It should be appreciated that the terms bacteria and bacterial strainsas used herein are interchangeable.

In some embodiments, the bacterial compositions disclosed herein can beused in therapeutic applications. In some embodiments, the bacterialcompositions disclosed herein can be used in pharmaceuticalcompositions. In some embodiments, the bacterial compositions used intherapeutic applications and/or pharmaceutical compositions, arebacterial compositions that were found to have integrity based on themethods provided herein (i.e., were not found to have a loss ofintegrity). In some embodiments, the solid compositions that includebacterial strains provided herein may be formulated for administrationas a pharmaceutical composition, e.g., by reconstitution of alyophilized product. It should be appreciated that a second aliquot ofthe bacterial compositions disclosed herein may be used in apharmaceutical composition. Thus, in some embodiments a first aliquot isused in the methods provided herein to evaluate the integrity of thebacterial composition. If the first aliquot of the bacterialcompositions was found to have integrity based on the methods providedherein (i.e., was not found to have a loss of integrity), a secondaliquot or additional aliquots of the bacterial composition may be usedin a pharmaceutical composition.

The term “pharmaceutical composition” as used herein means a productthat results from the mixing or combining of a solid formulationprovided herein and one or more pharmaceutically acceptable excipient.

An “acceptable” excipient refers to an excipient that must be compatiblewith the active ingredient (e.g., the bacterial strain) and notdeleterious to the subject to which it is administered. In someembodiments, the pharmaceutically acceptable excipient is selected basedon the intended route of administration of the composition, for examplea composition for oral or nasal administration may comprise a differentpharmaceutically acceptable excipient than a composition for rectaladministration. Examples of excipients include sterile water,physiological saline, solvent, a base material, an emulsifier, asuspending agent, a surfactant, a stabilizer, a flavoring agent, anaromatic, an excipient, a vehicle, a preservative, a binder, a diluent,a tonicity adjusting agent, a soothing agent, a bulking agent, adisintegrating agent, a buffer agent, a coating agent, a lubricant, acolorant, a sweetener, a thickening agent, and a solubilizer.

Strain 1 16S ribosomal RNA Clostridium bolteae (SEQ ID NO: 1)ATGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCAATTAAAATGAAGTTTTCGGATGGATTTTTGATTGACTGAGTGGCGGACGGGTGAGTAACGCGTGGATAACCTGCCTCACACTGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCACAGTACCGCATGGTACGGTGTGAAAAACTCCGGTGGTGTGAGATGGATCCGCGTCTGATTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTGAGAGGGTGACCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGTGAAGAAGTATTTCGGTATGTAAAGCTCTATCAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGCGAAGCAAGTCTGAAGTGAAAACCCAGGGCTCAACCCTGGGACTGCTTTGGAAACTGTTTTGCTAGAGTGTCGGAGAGGTAAGTGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGATAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTGGGGGGCAAAGCCCTTCGGTGCCGTCGCAAACGCAGTAAGCATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCCTCTTGACCGGCGTGTAACGGCGCCTTCCCTTCGGGGCAAGAGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTAGTAGCCAGCAGGTAAAGCTGGGCACTCTAGGGAGACTGCCAGGGATAACCTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCAAGACAGTGATGTGGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGTAGTCTGCAACCCGACTACACGAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGCAACGCCCGAAGTCAGTGACCCAACTCGCAAGAGAGGGAGCTGCCGAAGGCGGGGCAGGTAACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTStrain 2 16S ribosomal RNA Anaerotruncus colihominis (SEQ ID NO: 6)TCAAAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGCGCCTAACACATGCAAGTCGAACGGAGCTTACGTTTTGAAGTTTTCGGATGGATGAATGTAAGCTTAGTGGCGGACGGGTGAGTAACACGTGAGCAACCTGCCTTTCAGAGGGGGATAACAGCCGGAAACGGCTGCTAATACCGCATGATGTTGCGGGGGCACATGCCCCTGCAACCAAAGGAGCAATCCGCTGAAAGATGGGCTCGCGTCCGATTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCGGTAGCCGGACTGAGAGGTTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGAAGACGGTCTTCGGATTGTAAACCTCTGTCTTTGGGGAAGAAAATGACGGTACCCAAAGAGGAAGCTCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGAGCAAGCGTTGTCCGGAATTACTGGGTGTAAAGGGAGCGTAGGCGGGATGGCAAGTAGAATGTTAAATCCATCGGCTCAACCGGTGGCTGCGTTCTAAACTGCCGTTCTTGAGTGAAGTAGAGGCAGGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCCTGCTGGGCTTTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATTACTAGGTGTGGGGGGACTGACCCCTTCCGTGCCGCAGTTAACACAATAAGTAATCCACCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCGGATGCATAGCCTAGAGATAGGTGAAGCCCTTCGGGGCATCCAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATTAGTTGCTACGCAAGAGCACTCTAATGAGACTGCCGTTGACAAAACGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCACTAAAACAGAGGGCGGCGACACCGCGAGGTGAAGCGAATCCCGAAAAAGTGTCTCAGTTCAGATTGCAGGCTGCAACCCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTCGGTAACACCCGAAGCCAGTAGCCTAACCGCAAGGGGGGCGCTGTCGAAGGTGGGATTGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTStrain 3 16S ribosomal RNA Ruminococcus torques (SEQ ID NO: 3)TACGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAGCGAAGCGCTGTTTTCAGAATCTTCGGAGGAAGAGGACAGTGACTGAGCGGCGGACGGGTGAGTAACGCGTGGGCAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCACAGGACCGCATGGTGTAGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAGGTAGTTGGTGGGGTAAAGGCCTACCAAGCCGACGATCAGTAGCCGACCTGAGAGGGTGACCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATTTCGGTATGTAAACTTCTATCAGCAGGGAAGAAAATGACGGTACCTGAGTAAGAAGCACCGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGGTGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGATAGGCAAGTCTGGAGTGAAAACCCAGGGCTCAACCCTGGGACTGCTTTGGAAACTGCAGATCTGGAGTGCCGGAGAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTGACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGACTACTAGGTGTCGGTGTGCAAAGCACATCGGTGCCGCAGCAAACGCAATAAGTAGTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGGTCTTGACATCCGGATGACGGGCGAGTAATGTCGCCGTCCCTTCGGGGCGTCCGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTCAGTAGCCAGCATATAAGGTGGGCACTCTGGAGAGACTGCCAGGGAGAACCTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGGCCAGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGAGGGTGACCTGGAGCGAATCCCAAAAATAACGTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGCCAGTGACCCAACCTTAGAGGAGGGAGCTGTCGAAGGCGGGACGGATAACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTStrain 4 16S ribosomal RNA Clostridium symbiosum (SEQ ID NO: 4)ATGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGAAGCGATTTAACGGAAGTTTTCGGATGGAAGTTGAATTGACTGAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTTGTACTGGGGGACAACAGTTAGAAATGACTGCTAATACCGCATAAGCGCACAGTATCGCATGATACAGTGTGAAAAACTCCGGTGGTACAAGATGGACCCGCGTCTGATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGACGATCAGTAGCCGACCTGAGAGGGTGACCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGTGAAGAAGTATTTCGGTATGTAAAGCTCTATCAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGTAAAGCAAGTCTGAAGTGAAAGCCCGCGGCTCAACTGCGGGACTGCTTTGGAAACTGTTTAACTGGAGTGTCGGAGAGGTAAGTGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGACTTACTGGACGATAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTTGGGGAGCAAAGCTCTTCGGTGCCGTCGCAAACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCGATCCGACGGGGGAGTAACGTCCCCTTCCCTTCGGGGCGGAGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTCTAAGTAGCCAGCGGTTCGGCCGGGAACTCTTGGGAGACTGCCAGGGATAACCTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGATCTGGGCTACACACGTGCTACAATGGCGTAAACAAAGAGAAGCAAGACCGCGAGGTGGAGCAAATCTCAAAAATAACGTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCCAACCGCAAGGAGGGAGCTGCCGAAGGCGGGACCGATAACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTStrain 5 16S ribosomal RNA Blautia producta (SEQ ID NO: 5)ATCAGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACTTAAGTGGATCTCTTCGGATTGAAGCTTATTTGACTGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGGCTGCTAATACCGCATAAGCGCACAGGACCGCATGGTCTGGTGTGAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGCCTGAGAGGGTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAAGGAAGAAGTATCTCGGTATGTAAACTTCTATCAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGAAGAGCAAGTCTGATGTGAAAGGCTGGGGCTTAACCCCAGGACTGCATTGGAAACTGTTTTTCTAGAGTGCCGGAGAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGGTAACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCGGGTGGCAAAGCCATTCGGTGCCGCAGCAAACGCAATAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCCCTCTGACCGGCCCGTAACGGGGCCTTCCCTTCGGGGCAGAGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTAGTAGCCAGCAGGTGAAGCTGGGCACTCTAGGGAGACTGCCGGGGATAACCCGGAGGAAGGCGGGGACGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGCGTAAACAAAGGGAAGCGAGACAGCGATGTTGAGCAAATCCCAAAAATAACGTCCCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCCAACCTTACAGGAGGGAGCTGCCGAAGGCGGGACCGATAACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTStrain 6 16S ribosomal RNA Dorea longicatena (SEQ ID NO: 2)AACGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGAAGCACTTAAGTTTGATTCTTCGGATGAAGACTTTTGTGACTGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATACAGGGGGATAACAGTTAGAAATGACTGCTAATACCGCATAAGACCACGGTACCGCATGGTACAGTGGTAAAAACTCCGGTGGTATGAGATGGACCCGCGTCTGATTAGGTAGTTGGTGGGGTAACGGCCTACCAAGCCGACGATCAGTAGCCGACCTGAGAGGGTGACCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGAGGAAACTCTGATGCAGCGACGCCGCGTGAAGGATGAAGTATTTCGGTATGTAAACTTCTATCAGCAGGGAAGAAAATGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGAGCGTAGACGGCACGGCAAGCCAGATGTGAAAGCCCGGGGCTCAACCCCGGGACTGCATTTGGAACTGCTGAGCTAGAGTGTCGGAGAGGCAAGTGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTGCTGGACGATGACTGACGTTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGACTGCTAGGTGTCGGGTGGCAAAGCCATTCGGTGCCGCAGCTAACGCAATAAGCAGTCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTGATCTTGACATCCCGATGACCGCTTCGTAATGGAAGCTTTTCTTCGGAACATCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTCAGTAGCCAGCAGGTTAAGCTGGGCACTCTGGAGAGACTGCCAGGGATAACCTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCAGGGCTACACACGTGCTACAATGGCGTAAACAAAGAGAAGCGAACTCGCGAGGGTAAGCAAATCTCAAAAATAACGTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGCAGATCAGAATGCTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTCAGTAACGCCCGAAGTCAGTGACCCAACCGTAAGGAGGGAGCTGCCGAAGGTGGGACCGATAACTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTTStrain 7 16S ribosomal RNA Erysipelotrichaceae bacterium (SEQ ID NO: 7)ATGGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCATGCCTAATACATGCAAGTCGAACGAAGTTTCGAGGAAGCTTGCTTCCAAAGAGACTTAGTGGCGAACGGGTGAGTAACACGTAGGTAACCTGCCCATGTGTCCGGGATAACTGCTGGAAACGGTAGCTAAAACCGGATAGGTATACAGAGCGCATGCTCAGTATATTAAAGCGCCCATCAAGGCGTGAACATGGATGGACCTGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCCCACCAAGGCGATGATGCGTAGCCGGCCTGAGAGGGTAAACGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATTTTCGTCAATGGGGGAAACCCTGAACGAGCAATGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAGCTCTGTTGTAAGTGAAGAACGGCTCATAGAGGAAATGCTATGGGAGTGACGGTAGCTTACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATCATTGGGCGTAAAGGGTGCGTAGGTGGCGTACTAAGTCTGTAGTAAAAGGCAATGGCTCAACCATTGTAAGCTATGGAAACTGGTATGCTGGAGTGCAGAAGAGGGCGATGGAATTCCATGTGTAGCGGTAAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGTCGCCTGGTCTGTAACTGACACTGAGGCACGAAAGCGTGGGGAGCAAATAGGATTAGATACCCTAGTAGTCCACGCCGTAAACGATGAGAACTAAGTGTTGGAGGAATTCAGTGCTGCAGTTAACGCAATAAGTTCTCCGCCTGGGGAGTATGCACGCAAGTGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGGAAACAAATACCCTAGAGATAGGGGGATAATTATGGATCACACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCGCATGTTACCAGCATCAAGTTGGGGACTCATGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGGCCTGGGCTACACACGTACTACAATGGCGGCCACAAAGAGCAGCGACACAGTGATGTGAAGCGAATCTCATAAAGGTCGTCTCAGTTCGGATTGAAGTCTGCAACTCGACTTCATGAAGTCGGAATCGCTAGTAATCGCAGATCAGCATGCTGCGGTGAATACGTTCTCGGGCCTTGTACACACCGCCCGTCAAACCATGGGAGTCAGTAATACCCGAAGCCGGTGGCATAACCGTAAGGAGTGAGCCGTCGAAGGTAGGACCGATGACTGGGGTTAAGTCGTAACAAGGTATCCCTACGGGAACGTGGGGATGGATCACCTCCTTTStrain 8 16S ribosomal RNA Subdoligranulum spp (SEQ ID NO: 8)TATTGAGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGGGTGCTCATGACGGAGGATTCGTCCAACGGATTGAGTTACCTAGTGGCGGACGGGTGAGTAACGCGTGAGGAACCTGCCTTGGAGAGGGGAATAACACTCCGAAAGGAGTGCTAATACCGCATGATGCAGTTGGGTCGCATGGCTCTGACTGCCAAAGATTTATCGCTCTGAGATGGCCTCGCGTCTGATTAGCTAGTAGGCGGGGTAACGGCCCACCTAGGCGACGATCAGTAGCCGGACTGAGAGGTTGACCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGGCAATGGGCGCAAGCCTGACCCAGCAACGCCGCGTGAAGGAAGAAGGCTTTCGGGTTGTAAACTTCTTTTGTCGGGGACGAAACAAATGACGGTACCCGACGAATAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTACTGGGTGTAAAGGGCGTGTAGGCGGGATTGCAAGTCAGATGTGAAAACTGGGGGCTCAACCTCCAGCCTGCATTTGAAACTGTAGTTCTTGAGTGCTGGAGAGGCAATCGGAATTCCGTGTGTAGCGGTGAAATGCGTAGATATACGGAGGAACACCAGTGGCGAAGGCGGATTGCTGGACAGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGGATACTAGGTGTGGGGGGTCTGACCCCCTCCGTGCCGCAGTTAACACAATAAGTATCCCACCTGGGGAGTACGATCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGGCTTGACATCCCACTAACGAAGCAGAGATGCATTAGGTGCCCTTCGGGGAAAGTGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCTACGCAAGAGCACTCTAGCGAGACTGCCGTTGACAAAACGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGTCCTGGGCCACACACGTACTACAATGGTGGTTAACAGAGGGAGGCAATACCGCGAGGTGGAGCAAATCCCTAAAAGCCATCCCAGTTCGGATTGCAGGCTGAAACCCGCCTGTATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTCGGGAACACCCGAAGTCCGTAGCCTAACCGCAAGGAGGGCGCGGCCGAAGGTGGGTTCGATAATTGGGGTGAAGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACCTCCTTT

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms hall include the singular. The methods andtechniques of the present disclosure are generally performed accordingto conventional methods well-known in the art. Generally, nomenclaturesused in connection with, and techniques of biochemistry, enzymology,molecular and cellular biology, microbiology, virology, cell or tissueculture, genetics and protein and nucleic chemistry described herein arethose well-known and commonly used in the art. The methods andtechniques of the present disclosure are generally performed accordingto conventional methods well known in the art and as described invarious general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference, in particular for the teaching that isreferenced hereinabove. However, the citation of any reference is notintended to be an admission that the reference is prior art.

EXAMPLES Example 1: Evaluating Bacterial Integrity Using Selective Media

Experiment 1: Dorea longicatena

A lyophilized culture of Dorea longicatena (Strain 6, with 16S RNASequence SEQ ID NO:2) was prepared by growing in HiVeg reinforcedclostridial media until early stage stationary phase, and thenexchanging with 8% sucrose, 1% yeast extract, 0.05% cysteine, 20 mMhistidine, pH7 via diafiltration. This was frozen at 1° C. per minutefollowed by lyophilization. The overall titer after lyophilization wasreduced by approximately 3 logs from the starting culture. Thelyophilized culture was reconstituted in PBS before use.

A frozen culture of Dorea longicatena was created by mixing 6 ml of alate log phase culture in PYG with 2 ml of 60% glycerol to a finalconcentration of 15% glycerol. This was frozen at −80° C. for at least 1week. The culture was thawed at room temperature before use.

A healthy fresh culture of Dorea longicatena was created by inoculatinga fresh colony into reduced PYG media and incubating at 37° C. inanaerobic conditions overnight.

Each of the above Dorea longicatena cultures were serially diluted inPBS. 100 ul of dilutions were plated in duplicate on pre-reducedTrypticase Soy Agar supplemented with 5% horse blood (TSBA) andpre-reduced TSAB with the addition of 0.15% Ox Bile (the bile acidmedia). Petri plates were incubated anaerobically for 3 days at 37° C.and then the colonies were counted and the titers calculated based onthe dilutions and plated volume. The results are shown in FIGS. 1A and1B.

The PYG control data were generated using a BioscreenC assay: 50 ul ofdilutions were added to 350 ul reduced PYG in wells of a BioscreenCgrowth curve analyzer. For the fresh culture, four replicates of the10⁻² and 10⁻⁴ dilutions, five replicates of the 10⁻⁵ and 10⁻⁶, twentyreplicates of 10⁻⁷, thirty replicates of 10⁻⁸ and thirty eightreplicates of 10⁻⁹ were used. No growth was observed in the 10⁻⁹dilution. For the lyophilized culture, four replicates of the 10⁻² and10⁻⁴ dilutions, ten replicates of 10⁻⁵, thirty replicates of 10⁻⁶, andforty replicates of 10⁻⁷ were used.

Using the lowest three dilutions that showed growth (10⁻⁶, 10⁻⁷, and10⁻⁸ for fresh, and 10⁻⁵, 10⁻⁶, and 10⁻⁷ for lyophilized), the titer wasestimated using a most probable number calculation, solving for Xiteratively from the following equation:

${\sum\limits_{j = 1}^{k}\frac{g_{j}m_{j}}{1 - {\exp \left( {{- \lambda}\; m_{j}} \right)}}} = {\sum\limits_{j = 1}^{k}{t_{j}m_{j}}}$

where exp(x) means e′, and

K denotes the number of dilutions,

g_(j) denotes the number of positive (or growth) weds in the jthdilution,

m_(j) denotes the amount of the original sample put in each well in thejth dilution,

t_(j) denotes the number of we in the jth dilution.

The results are shown in FIGS. 1A and 1B.

Experiment 2: Clostridium bolteae

A lyophilized culture of Clostridium bolteae (Strain 1, with 16S RNASequence SEQ ID NO:1) was prepared by growing in Hi Veg reinforcedclostridial media until early stage stationary phase, and thenexchanging with 7.5% Trehalose, 5% Poloxamer, 1% yeast extract, 0.05%cysteine, 20 mM histidine, pH7 via diafiltration. This was frozen at 1°C. per minute followed by lyophilization. The overall titer afterlyophilization was reduced by greater than 2 logs from the startingculture. The lyophilized culture was reconstituted in PBS before use.

A healthy fresh culture of Clostridium bolteae was created byinoculating a fresh colony into reduced PYG media and incubating at 37°C. in anaerobic conditions overnight.

Each of the above Clostridium bolteae cultures were serially diluted inPBS. 100 μl of each of the dilutions were plated in duplicate onpre-reduced Trypticase Soy Agar supplemented with 5% horse blood (TSBA)and pre-reduced TSAB with the addition of 0.15% Ox Bile (the bile acidmedia). Petri plates were incubated for 3 days at 37° C., and then thecolonies were counted and the titers calculated based on the dilutionsand plated volume. The results are shown in FIGS. 2A and 2B.

Example 2: Evaluating Bacterial Integrity Using Selective MediaFollowing Lyophilization

Viability of bacterial compositions was assessed prior to and afterlyophilization and storage. In preparing bacterial compositions, forexample for use in pharmaceutical compositions for therapeuticapplications, the bacterial compositions are cultured and subjected to alyophilization step, then stored at −80° C., which may impact theviability of the cells. The viability of bacterial compositions prior toand after lyophilization was assessed to determine whether preparationfor the lyophilized composition results in a difference in the viabilitycompared to the bacterial compositions prior to lyophilization. Platingthe bacterial strains on Ox Bile measures the viability under a stresscondition, exposure to bile salts.

The viability of the bacterial strains (Strains 1-8) on non-selectivemedia (Chocolate Agar) and selective media (Chocolate Agar plus Ox Bile)prior to lyophilization is presented in FIG. 3A, and the viability ofthe bacterial strains (Strains 1-8) on non-selective media (ChocolateAgar) and selective media (Chocolate Agar plus Ox Bile) followinglyophilization is presented in FIG. 3B.

Example 3: Evaluating Bacterial Integrity Using Selective MediaFollowing Storage

Viability of bacterial compositions was assessed prior to and afterstorage at 25° C. Bacterial compositions, for example in pharmaceuticalcompositions for therapeutic applications, may be stored, for example at25° C. (room temperature) prior to administration to a subject, whichmay impact the viability of the cells. The viability of bacterialcompositions that had been lyophilized was assessed after variouslengths of time at 25° C.

FIG. 4A shows viability of bacterial Strain 1 (Clostridium bolteae) andStrain 6 (Dorea longicatena) on non-selective media and selective media(containing Ox Bile) following storage for 48 hours or one week at 25°C. FIG. 4B shows viability of bacterial Strain 1 (Clostridium bolteae)and Strain 6 (Dorea longicatena) on non-selective media and selectivemedia (containing Ox Bile) following storage for one month at 25° C.

1. A method for determining the integrity of a bacterial composition,the method comprising: growing the bacterial composition on a selectivemedium, wherein the bacterial composition comprises a bacterial strain,and wherein if the bacterial composition grows slower on the selectivemedium than on a non-selective medium, the integrity of the bacterialcomposition is compromised.
 2. The method of claim 1, further comprisinggrowing the bacterial composition on the non-selective medium.
 3. Themethod of claim 1, wherein the bacterial composition was lyophilizedprior to growing the bacterial composition.
 4. The method of claim 1,wherein if the integrity of the bacterial composition is compromised,the bacterial composition is not used to prepare a live bacterialproduct.
 5. A method for determining the integrity of a first bacterialcomposition, the method comprising: growing the first bacterialcomposition on a selective medium, wherein the first bacterialcomposition comprises a bacterial strain; growing a second bacterialcomposition on the selective medium, wherein the second bacterialcomposition comprises a bacterial strain, and wherein if the firstbacterial composition grows slower than the second bacterialcomposition, the integrity of the first bacterial composition iscompromised.
 6. A method for determining the integrity of a firstbacterial composition, the method comprising: growing the firstbacterial composition on a selective medium, growing the first bacterialcomposition on a non-selective medium, wherein the first bacterialcomposition comprises a bacterial strain; growing a second bacterialcomposition on the selective medium, growing the second bacterialcomposition on the non-selective medium, wherein the second bacterialcomposition comprises a bacterial strain; wherein if the difference ingrowth between the selective medium and the non-selective medium for thefirst bacterial composition is greater than the difference in growthbetween the selective medium and the non-selective medium for the secondbacterial composition, and both the first bacterial composition and thesecond bacterial composition grow slower on the selective medium thanthe non-selective medium, the integrity of the first bacterialcomposition is compromised.
 7. The method of claim 5, wherein the firstbacterial composition was lyophilized prior to growing the firstbacterial composition.
 8. The method of claim 5, wherein the secondbacterial composition was lyophilized prior to growing the secondbacterial composition.
 9. The method of claim 1, wherein the selectivemedium is a bile acid media.
 10. The method of claim 5, wherein thefirst bacterial composition and the second bacterial compositioncomprise the same bacterial strain.
 11. The method of claim 5, whereinif the integrity of the first bacterial composition is compromised, thefirst bacterial composition is not used to prepare a live bacterialproduct.
 12. The method of claim 1, wherein the bacterial strain is ananaerobic bacterial strain.
 13. The method of claim 1, wherein thebacterial strain belongs to the class Clostridia.
 14. The method ofclaim 1, wherein the bacterial strain belongs to the familyClostridiaceae.
 15. The method of claim 1, wherein the bacterial strainbelongs to the genus Clostridium.
 16. The method of claim 1, wherein thebacterial strain is selected from the group consisting of Clostridiumbolteae, Anaerotruncus colihominis, Ruminococcus torques, Clostridiumsymbiosum, Blautia producta, Dorea longicatena, Erysipelotrichaceaebacterium, and Subdolinogranulum spp.
 17. The method of claim 16,wherein the bacterial strain is Clostridium bolteae.
 18. The method ofclaim 16, wherein the bacterial strain is Dorea longicatena.
 19. Themethod of claim 1, wherein the bacterial strain comprises a 16S rDNAsequence having at least 97% sequence identity with a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 1-8.